The present invention relates to a diagnostic apparatus having utility in the radioimmunoassay of antigens and their antibodies. More particularly, the invention relates to an apparatus for a two-site immunoradiometric assay for serum ferritin in human blood samples.
Ferritin is a high molecular weight protein functioning primarily as a situs for iron storage. Ferritin can be found in almost every solid tissue of the human body, but it appears principally in the cytoplasm of hepatic and reticuloendothelial cells. Prior to about 1972, it had been assumed that ferritin only existed in extracellular fluid under abnormal conditions. It has since been discovered that the concentration of serum ferritin directly correlates with the content of iron in body stores in normal human beings. Thus, the measurement of serum ferritin levels provides an effective method by which the status of body iron stores may be evaluated and clinical diagnoses made therefrom. For example, an accurate measurement of the serum ferritin level will enable a clinician to differentiate between that anemia caused by iron depletion and other forms of anemia.
Inasmuch as ferritin is an antigenically active macromolecule, the presence of ferritin can be detected by techniques which have been developed in the past for detecting various other antigenic materials. The radioimmunoassay procedure is an example of such a technique.
Addison et al, Journal of Clinical Pathology, Volume 25, pages 326-329 (1972) disclose a technique wherein human blood serum is treated with soluble purified radioactive ferritin antibodies. The radioactive complex produced remains in solution while unused radioactive antibodies, i.e., those which do not link up with the antigen, are removed by a second reaction with a solid phase antigen.
Miles et al, Analytical Biochemistry, Volume 61, pages 209-224 (1974) suggests an improvement in this technique wherein the ferritin is first insolubilized and thereafter is made to react with soluble labeled antibody. The labeled complex is thus insoluble and any unreacted labeled antibody can be washed away. It will be appreciated that the amount of ferritin present will be directly proportional to the radioactivity in solid phase.
Solid phase radioimmunoassay of antigens is believed to have been developed by Catt (see, e.g., U.S. Pat. No. 3,646,346 to Catt) and others (see for example, U.S. Pat. No. 3,790,663 to Garrison et al). Basically, the prior art teaches the solid phase antibodies are coated on a polystyrene substrate. The substrates are then placed in contact with serum containing ferritin and permitted to stand for a period of time to enable reaction to occur. After an incubation period, the substrates are washed, dried, and a radioactive count measurement is taken. The measurement is compared with standard values for different serum ferritin concentration levels.
More recently, a two-site immunoradiometric assay for serum ferritin has been developed. This new test is basically a two stage reaction. In the first stage, human serum ferritin is bonded to a solid-phase anti-human ferritin. In the second stage, a purified, radiolabeled anti-human ferritin is bound to the first stage reaction product. Then the solid phase is washed and counted in a radiation counter. The concentration of serum ferritin may be calculated by comparing the unknown with a simultaneously run standard sample.
Apparatus for performing a quantitative radioimmunoassay have been somewhat varied. The present invention is directed to an improvement over apparatus which includes the following components. Firstly there is provided a water insoluble polymeric substrate fashioned as a sphere or bead. This bead is coated with a previously formed human ferritin antibody. After the beads have been prepared, the beads are washed with deionized water.
Later, each bead is washed in a container such as a beaker. After the washing is completed, the container is aspirated to remove the rinsing water. Immediately after aspiration of the water, a bead would have to be placed in a test tube having a diluted sample of patient serum and a test tube containing the test standard. The beads would remain in the test tubes at room temperature for six hours or for sixteen hours at 4.degree. C. Alternatively, the test tubes could be covered and placed on a horizontal rotating table and shaken for approximately two hours at a moderate speed at room temperature.
In the prior art, after incubation, the reaction solution is aspirated from the test tube bearing the patient sample and the test tube bearing the standard. Then each bead is washed twice with a special washing solution. This washing solution is sprayed onto the bead and is almost immediately thereafter aspirated by an apparatus especially adapted for such use. Each bead is washed twice with approximately one minute between washings. The test tubes must be aspirated twice to remove any liquid that may have drained from the sides of the test tubes.
After washing and aspirating, the radiolabeled antibody is introduced onto the beads in the test tubes. The test tubes are then refrigerated at 4.degree. C. for sixteen hours. Alternatively, the test tubes can be covered and shaken on a horizontal shaking apparatus for 2 hours at a moderate speed at room temperature.
After this second incubation, the radioactive solution is aspirated from each test tube utilizing a special apparatus. This relatively complex device is used to wash each bead three times with a washing solution. The washing solution, of course, must be aspirated after each wash. After the last wash, the test tubes are required to remain stationary for an additional minute to give liquid on the side of the test tube an opportunity to drain to the bottom. This additional fluid should be aspirated.
Each of the beads is then removed from their respective test tubes and introduced into special counting tubes which were not used in the assay. The counting tubes are introduced into a gramma radiation counter set to detect the labeling isotope. The beads are counted for a time appropriate for adequate statistical significance, and the appropriate calculations are made in a now well-known manner to provide a quantitative determination of iron ferritin in the patient.
While such arrangements have exhibited at least a degree of utility in radioimmunoassay, room for significant improvement remains. The beads utilized in at least some known iron ferritin analyses are 1/4" in diameter and are relatively difficult to handle. Handling by a pathologist or laboratory technician introduces the risk of contamination both to the test sample and to the personnel. Because the 1/4" diameter beads are difficult to handle, they may be dropped thus necessitating repeating the time-consuming first stage and second stage incubation periods. This time delay may have adverse consequences to the patient and results generally in an inefficient laboratory operation.
Utilizing known apparatus, the beads would require handling after both the first and second phase incubations have occurred. If a test sample were spoiled at the second phase of the procedure, several days may be required to repeat the procedure.
Of independent significance, is the relatively complex equipment required to wash and aspirate each test tube containing a test bead. In order to achieve satisfactory washing, the washing procedure must be repeated once and is preferably repeated another time. Aspiration is required between washings, and both a pressure pump and a suction pump are required. The pressure pump is required to introduce the washing solution into each test tube, and a suction pump is required to draw the washing solution out of the test tube through a specially adapted conduit. It will be appreciated that in the event of a failure of the relatively expensive washing or aspirating equipment, the assay might be terminated or spoiled. In addition, it is believed that with washing and aspirating, even three times and with a specially formulated washing solution, there may not be an adequate washing of each bead.
The problems enumerated in the foregoing are not intended to be exhaustive but rather are among many which tend to impair the effectiveness of previously known radioimmunoassay apparatus. Other noteworthy problems may also exist; however, those presented above should be sufficient to demonstrate that radioimmunoassay apparatus appearing in the art have not been altogether satisfactory.